The molecular networks mediated by histone lysine methylation are intricate and extremely complex. Currently, the action mechanisms of the signaling pathways mediated by histone lysine methylation are still very elusive. It is hypothesized that the histone methylation at different lysine residues and at different states (e.g., di- and trimethylation) are recognized by the corresponding binding effectors that subsequently turn on and off the related downstream signaling pathways. One major problem that remains to be addressed is how to define the methylation-dependent interactome for each methylated histone tail. This project is aimed at developing transforming technologies that allow the identification of the natural repertoire of the effector proteins that bind to each of the methylated histone states at lysine residues. Powerful techniques at the interface of chemistry and biology are integrated to address the problem. Specifically, we will use a novel proteomic technique called mRNA-display in which each protein is covalently linked to its own mRNA. The protein sequences that bind to distinct methylation states will be isolated from an mRNA-displayed human proteome library. The selections will be performed under very specific conditions. After capturing the methylated histone tail/binding partner complexes, the methylation-independent binders will be removed by using the unmodified histone peptide, while those binding partners that are dependent on the desired methylation state will be specifically eluted using the corresponding di- or trimethylated histone peptide. Due to the conjugation between the genotype and the phenotype, the selected sequences can be readily amplified for iterative rounds of selection, allowing us to address the difficulty in identifying the relatively weak methylation-dependent protein-protein interactions on a proteome wide scale. As a backup plan, we will use the methylated histone tail peptides that contain a crosslinkable photo-leucine moiety to facilitate the capture of low abundant proteins whose interactions with methylated histone tails are relatively weak. Although we address the problem using the human proteome in this project, the method can be readily applied to model organisms such as C. elegans, zebrafish and Drosophila. PUBLIC HEALTH RELEVANCE: The availability of the methylation-dependent interactome will allow us to decipher the binding effectors of each histone methylation state and therefore greatly facilitate our understanding of the signaling pathways mediated by histone lysine methylation. It should also aid in the development of therapeutic and imaging agents that allow the manipulation of epigenetic processes involved in a number of human diseases including cancer.